For the microscopic optical examination of samples, various measurement methods exist which provide different kinds of information. Either image or point measurements can be performed. In image measurements, generally, the value of a physical quantity is recorded in dependence on the measurement site. The measurement of this physical quantity is performed either at numerous different points or measurement sites in parallel, or respectively only at one point, and the site of this point, i.e. the measurement site, will be varied. The latter is often referred to as a “screening” of the sample surface. In this case, each individual measurement has to be performed very fast so that the image measurement can be concluded within an acceptable length of time.
Examples of useful image measurement methods are: bright-field, dark-field, total-internal-reflection, fluorescence, 2-photon-fluorescence, fluorescence-lifetime, fluorescence-emission-spectroscopy, polarization and fluorescence-polarization microscopy. Further, these methods can be performed by use of different detectors such as e.g. line cameras or surface cameras, and, in part, these detectors can be used both in conventional and confocal arrangements.
In point measurements, contrary to image measurement, the measurement is carried out only at one site on the sample, or the site information is not evaluated, or an averaging including a large number of measurement sites is performed (conventional measurement by use of a large-surfaced detector). Correspondingly, the individual measurement may be both more complex and more time-consuming. The complexity can relate both to the measurement apparatus and the data obtained (e.g. complete spectra). For instance, in the fluorescence correlation spectroscopy (FCS) point measurement method, the fluctuation of a fluorescence signal coming from a small volume is recorded over a longer period of time, and the signal is used to derive information on photophysical, chemical and physical properties of fluorescent particles and molecule in this volume. In these measurements, it is often assumed beforehand that the volume observed is representative of the sample. This will of course largely apply to homogenous samples but only to a restricted extent if also structured components occur in the sample. Useful point measurement methods are—although the following examples are not exhaustive—FCS, FIDA, fluorescence-emission, fluorescence-excitation and fluorescence-absorption spectroscopy, fluorescence-lifetime spectroscopy and fluorescence-anisotropy measurements, as described in numerous scientific and technical publications. The measurements can often be performed both in a conventional and a confocal arrangement.
For the characterization of substances with regard to possible pharmaceutical and medical uses, it is often reasonable and necessary to carry out an examination by use of image and point measurement methods alike. Then, information on all components can be extracted from samples containing homogeneous as well as structured components. By way of example, it could be examined in what manner a sample comprising cells which are assumed to release certain molecules into the solution surrounding them, will react on an added test substance. With the FCS method, the possible occurrence of a biochemical reaction in the solution can be detected while fluorescence microscopy makes it possible to examine the cell layer and to obtain indications e.g. of a possible toxic nature of the test substance.
In multi-channel microscopy, a sample can be subjected to imaging measurement methods for performing a plurality of measurements of the same sample region under different measurement conditions or with different detectors. Thereby, for instance, there is determined the distribution of different fluorophobes in the sample, which differ from each other with regard to their photophysical properties such as e.g. the excitation and emission spectra and/or the fluorescence lifetime.
As to the devices wherein image measurement methods are combined with point measurement methods, mention should be made of the FCS apparatus for intracellular FCS by Brock (see e.g. Brock, “Fluorescence Correlation Microscopy and Quantitative Microsphere Recruitment Assay”, dissertation, 1999). This apparatus comprises a fluorescence microscope which has been retrofitted to include the components required for FCS. The selection among the measurement methods of microscopy or FCS is realized by a hinged mirror arranged in the path of rays.
To begin with, such a mechanical switching of the path of rays by means of a hinged mirror entails the necessity to determine the positions of the different measurement volumes relative to each other. High-throughput applications are subject to the additional necessity to perform this switching cycle a large number of times, thus causing mechanical wear of the constructional components. This wear process particularly affects a predefined relative position of the measurement volumes so that this position would have to be redefined at regular intervals. In certain cases, even the complete switching unit has to be replaced because of wear. Especially in high-throughput applications, a disadvantage is caused by the loss of measurement time due to the switching process, resulting in a decrease of the obtainable sample throughput.
So-called laser scanning microscopes (LSM) generate a two-dimensional image of a sample by sequential screening of the object plane with a single small measurement volume. Based on the knowledge on the position of the measurement volume at each point of time and on the corresponding picked-up measurement value, an image of this plane can be generated. Possible signals for this purpose include reflection and fluorescence intensity but also anisotropy and fluorescence anisotropy. In microscopes of the above type, a combination with point measurement methods such as e.g. the fluorescence emission spectroscopy is relatively simple since only the movement of the measurement volume need be eliminated for obtaining a sufficient measurement time.
When using laser scanning microscopes, a disadvantage resides in the relatively long measurement times for the individual images. A use of such microscopes for high-throughput screening will thus not be possible because the required short measurement times with sufficient signal quality cannot be obtained. In short measurement times, the measurement results will have only a small signal-to-noise ratio, so that an averaging has to be performed over several images. This averaging is carried out for improving the signal-to-noise ratio and therefore is necessary in case of short measurement times for an individual image. In case of long measurement times, a sufficient signal-to-noise ratio can be obtained also without averaging. This in turn will increase the measurement time. Further, the laser scanning microscope is restricted to the use of relatively complex image-generating methods.
To be able to distinguish a plurality of fluorophores in a microscope on the basis of their excitation and emission spectra, special optical filters, chromatic cameras and imaging spectrographs are used in multi-channel microscopes.
Normally, when optical filters are used, at least three filters will be employed. The first filter determines the excitation wavelength range, the second filter is a dichromatic mirror which reflects the excitation light and transmits the emission light (or vice versa). In front of the detector, a further filter is inserted, transmitting only the emission range. For the detection of different fluorophores, the possibility exists to exchange all three filters, according to the standardized routine when using commercially available microscopes. Alternatively, a single filter set can be arranged for a plurality of colorants if the excitation and emission wavelengths are sufficiently remote from each other, as is the case e.g. for the colorants DAPI, FITC and TRITC. If these colorants have to be recorded separately, a color film or a color CCD camera can be used. Likewise suited is a monochrome camera in combination with a selectable excitation spectrum, as performed e.g. in the so-called Pinkel filter sets. Filter sets also offer the opportunity to distinguish colorants from each other on the basis of their Stokes shift. In this manner, different colorants can be excited by the same wavelength and will differ from each other by the different displacement of the emission spectrum.
In multi-channel microscopes, it is frequently required to exchange one or several filters between the individual measurements. When using high-throughput systems, this causes considerable disadvantages, such as e.g. wear of components or losses in measurement time due to the switching times. Systems adapted to detect a plurality of colorants simultaneously are restricted to mere few combinations of colorants because the emission spectra must be sufficiently different and must not overlap with the excitation spectra of the other colorants. Further, the manufacture of such optical filters which are suited for a plurality of colorants is very complex so that a change towards other colorants is impeded by the considerable expenses for development and manufacture. This imposes heavy restrictions on the selection of the possible colorants. This disadvantage affects also systems which are based on spectrographs since also these require that the excitation light be attenuated. Further, imaging spectrographs are not particularly suited to record an image of the sample because the recording times—and possibly also the processing times—are frequently as long as several seconds.
It is an object of the invention to provide a device and a method for the measurement of chemical and/or biological samples which make it possible to perform different measurements of a sample, particularly through an image measurement method and a point measurement method, in a simple manner.